It is known that hypoxia-inducible factor 1 (HIF-1) activity results in the coordinated up-regulation of a large number of proteins that facilitate cell survival in tumours; however, the effect of HIF-1 on cancer metabolism is less well characterised. With knowledge of the specific effect of HIF-1 on cancer metabolism, biomarkers could be identified for which new drugs could be targeted. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) offers the potential to analyse intact cells in situ and has a mass spectral coverage that is applicable to metabolic profiling. It has been used to analyse the effects of HIF-1 on multicellular tumour models. Multicellular tumour spheroids (MTSs) have been cultured from human colon carcinoma cells with and without the expression of HIF-1, and the surface of the cross sections of each MTS has been analysed. Because metabolic profiling is an emerging field in ToF-SIMS, there is a requirement to determine which metabolites can be detected using this technique and which of those can be identified in complex mixtures within biological samples. For this, a selection of metabolites have been analysed, and the ToF-SIMS standard spectra acquired have been used to localise metabolites in MTS sections. The comparison of metabolic profiles of MTSs with and without the expression of HIF-1 has elucidated potential biomarkers for tumour survival in hypoxia, some of which may be HIF-1 regulated. Copyright © 2012 John Wiley & Sons, Ltd.